Predicting hepatotoxicity using cell based assays

ABSTRACT

Cell based assays are used to assess the hepatotoxicity of a stimulus. Imaging technologies are used to analyze the effects of a stimulus on hepatocytes. Image analysis may characterize the stimulus on the basis of whether it is hepatotoxic, and if so what type of pathology is exhibited; e.g., apoptosis, necrosis, cholestasis, and/or steatosis.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation-in-part, claiming priority under 35U.S.C. § 120, from U.S. co-pending patent application Ser. No.10/623,486 by Mattheakis et al., filed Jul. 18, 2003 and titled“PREDICTING HEPATOTOXICITY USING CELL BASED ASSAYS.” That application isincorporated herein by reference for all purposes.

BACKGROUND

I. Field of the Invention

The present invention relates to methods and apparatus for assessing thehepatotoxicity of a stimulus. More particularly, the present inventionrelates to image analysis methods and apparatus that characterize astimulus based upon phenotypic characteristics of hepatocytes and someother cell types exposed to the stimulus.

II. Background

Hepatotoxicity is a major safety concern for drug development.Approximately 90 percent of lead candidates fail to become drugs, andhepatotoxicity accounts for about 22 percent of these failures.Traditionally, a variety of strategies have been used to predicthepatotoxicity during preclinical development. These include incubatingcompounds with cultured hepatocytes to measure cytotoxicity or inductionof the various isoforms comprising the drug metabolizing CYP enzymes.Biochemical enzyme assays, using purified CYP enzymes or crude livermicrosome extracts, are used to determine the substrate activities ofdrug candidates and to profile their metabolic products usingchromatographic methods.

Animal studies have also been widely used to predict humanhepatotoxicity. In these studies, rats or mice are dosed with variousconcentrations of the test compound, and the animals are monitored forimportant serum markers such as serum albumin, prothrombin, bilirubin,AST, ALT, and alkaline phosphate at different time points. The animalsare then sacrificed, and a full histopathological analysis of the liver,kidney, and other important organs and/or tissues is carried out.

More recently, gene expression studies have been used to predicthepatotoxicity. The RNA is isolated from cultured hepatocytes or liversections from animals and analyzed using microarray technology. Theadvantages of this approach include faster turnaround times and lesslabor compared to animal pathology studies, and the method (as appliedto cultured hepatocytes) requires a smaller amount of the experimentalcompound. Many industrial and academic groups are attempting to identifykey genes that are expressed during a hepatotoxic response. The goal isto create a database that contains the gene expression patterns of knownhepatotoxins and associated liver pathologies. The database is then usedto predict the mechanism of hepatotoxicity by comparing the geneexpression patterns of a new compound to those of reference compounds.This approach is still under development, and the number of marker genesreported to be relevant for the rat model varies from 400 to over 3000genes. See “Serious liver injury: leading reasons for drug removals,restrictions” www.fda.gov/fdac/features/2001/301_liver.html; andToxExpress™ Application Note, GeneExpress ToxExpress Predictive System™,Gene Logic, Gaithersburg, Md. (2002).

Unfortunately, none of the traditional approaches adequately predictsthe hepatotoxic potential of drugs that reach the marketplace. At leastthree drugs within the last five years, Duract (bromfenac), Trovan(trovafloxacin) and Rezulin (troglitazone), have significant uselimitations or were pulled from the market due to human hepatotoxicity.Thus, there is a great need for new methods to predict hepatotoxicity,and to use these methods early in the lead optimization process to savetime and cost.

SUMMARY

The techniques of the present invention address the above need byproviding methods, program instructions and apparatus that assess thetoxicity of a stimulus. The invention accomplishes this by using imagingtechnologies to analyze the effects of a stimulus on hepatocytes orother cell types.

One aspect of the invention provides methods of assessing thehepatotoxicity of a stimulus, by performing the following operations:(a) exposing a hepatocyte culture to the stimulus; (b) imaging thehepatocytes; (c) analyzing an image of the hepatocytes to extractfeatures characterizing the hepatocytes; and (d) classifying thestimulus by quantitatively evaluating the extracted features to identifyone or more hepatotoxic pathologies resulting from the stimulus.Examples of hepatotoxic pathology classifications include necrosis,cholestasis, steatosis, fibrosis, apoptosis, and cirrhosis.

Another aspect of the invention provides methods of identifying anecrotic hepatotoxic pathology resulting from a stimulus. Such methodsmay be characterized by the following operations: (a) exposing ahepatocyte culture to the stimulus; (b) contacting the hepatocyteculture with markers relevant to necrosis (e.g., esterase activity andcell membrane permeability); (c) imaging the hepatocyte culture; (d)analyzing images of the hepactocyte culture to extract features relevantto necrosis; and (e) characterizing the necrotic response of thehepatocyte culture to the stimulus based on the extracted features. Inone embodiment, the method identifies average levels of esteraseactivity and cell membrane permeability for the hepatocyte culture basedon the extracted features. This information is then used tocharacterizing the necrotic response.

Still another aspect of the invention pertains to methods of identifyingan apoptotic hepatotoxic pathology resulting from a stimulus. Suchmethods may be characterized by the following operations: (a) exposing ahepatocyte culture to the stimulus; (b) treating the hepatocyte cultureunder conditions that distinguish apoptotic and non-apoptotichepatocytes (e.g., washing, exposing to markers for enzyme activity,exposure to markers for DNA); (c) imaging the hepatocyte culture; (d)analyzing images of the hepactocyte culture to extract features relevantto apoptosis; and (e) characterizing the apoptotic response of thehepatocyte culture to the stimulus based on the extracted features.

In a specific embodiment, the method includes (a) exposing a first andsecond hepatocyte culture to the stimulus; (b) performing a single steppreparatory treatment of the first hepatocyte culture, wherein thesingle step preparatory treatment does not include washing; (c)performing a multi-step preparatory treatment of the second hepatocyteculture, wherein the multi-step preparatory treatment includes washing;(d) imaging the first and second hepatocyte cultures; (e) analyzingimages of the first and second hepactocyte cultures to extract featuresrelevant to apoptosis; and (f) characterizing the apoptotic response ofthe hepatocytes to the stimulus based on the extracted features. Themethod may use to the extracted features to, for example, identifycondensation of the nuclei, cell adhesion, and average caspase-3activity for the first and second hepatocyte cultures. This allows themethod to characterize the apoptotic response of the first and secondhepatocyte cultures to the stimulus based on the characteristics of thenuclei, cell adhesion, and average caspase-3 activity.

Yet another aspect of the invention pertains to computer programproducts including machine-readable media on which are stored programinstructions for implementing a portion of or an entire method asdescribed above. Any of the methods of this invention may berepresented, in whole or in part, as program instructions that can beprovided on such computer readable media. In addition, the inventionpertains to various combinations of data generated and/or used asdescribed herein. Examples include databases, data structures, andlinked lists.

These and other features and advantages of the present invention will bedescribed in more detail below with reference to the associated figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cartoon representation of normal hepatocytes, showingvarious features as they might appear in the absence of a toxicresponse.

FIG. 1B is a flow chart depicting various operations commonly performedtogether in one implementation of a hepatotoxicity analysis of thisinvention.

FIG. 2 is a diagrammatic representation of hepatocytes exhibitingdrug-induced apoptosis.

FIG. 3 is a flow chart depicting various operations commonly performedtogether in one implementation of a hepatotoxicity analysis configuredto classify cells as apoptotic or necrotic.

FIG. 4A is a diagrammatic representation of hepatocytes exhibitingmicrovesicular steatosis.

FIG. 4B is a diagrammatic representation of hepatocytes exhibitingmacrovesicular steatosis.

FIG. 5 is a diagrammatic representation of a computer system that can beused with the methods and apparatus of the present invention.

FIG. 6 is a diagrammatic representation of sample compound and controlwell assignments for an assay block used in an example.

FIGS. 7A and 7B are tables showing experimental results for ahepatotoxicity assay conducted in accordance with this invention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

I. Introduction and Overview

Hepatotoxicity is a major safety concern for drug development. Asexplained above, a variety of strategies traditionally have been used topredict hepatotoxicity during preclinical development. As indicated, theexisting techniques have serious limitations and in fact some drugs haverecently been banned or significantly limited in use due to latediscovery of human hepatotoxicity. Accordingly, there is a need forbetter methods to predict human hepatotoxicity earlier in the drugdevelopment process.

This invention employs image and data analysis technology to provide anindication of whether a particular compound or other stimulus ishepatotoxic. Additionally, it may classify hepatotoxicity by thespecific pathologies. This detailed classification can be beneficial fordrug development in a number of ways.

First, knowing the exact pathology caused by a given compound can helpto determine whether the compound should be abandoned as a drugcandidate or whether it might be redesigned (e.g., subjected to a slightmolecular change) to reduce its liabilities. If modification is likelyto be successful, identification of the particular pathology elicitedcan also provide insight about which specific modifications are likelyto reduce toxicity.

In addition, knowing the pathology associated with a given stimulus canhelp to determine the nature of the risk associated with the stimulus.In general, a stimulus that exhibits some level of cytotoxicity may ormay not represent a low overall risk to a person's health. But knowingthe particular pathology caused by a given stimulus can provide morespecific information about the level of risk presented by the stimulus.For instance, some pathologies are only problematic or otherwiserelevant when the condition to be treated is a chronic condition, e.g.,fribrosis or cirrhosis of the liver. Other pathologies are onlyproblematic for acute conditions, where the compound under investigationis administered over a relatively short period of time, possibly in highdoses.

As explained further below, the invention extends to toxicity andpathologies in a wide range of systems (e.g., cell types, cell lines,tissues, etc.) other then hepatocyte systems. Examples include toxicityin dermal cells, kidney cells, neurons, etc. For convenience, theinvention will be described primarily in terms of hepatotoxicity.

In accordance with this invention, toxicity may be detected by image anddata analysis in various ways. Most conservatively, any effect to a cellthat is not considered the principal action of the stimulus of interestis viewed as an, off-target effect, or effect is viewed as an indicatorof potential toxicity. More specific assays consider particular indiciaof toxicity or a particular pathology. One such assay considersincreased level of cell necrosis in wells exposed to the stimulus inquestion. Other assays consider indicia of specific pathologies such ascholestasis or steatosis.

Regarding off-target effects, most stimuli, particularly those involvingexposure to drug or drug candidates, are evaluated for their effect on atarget (i.e., a protein or other component of the cell that isimplicated in a disease pathway). “On-target” effects are those effectsof the stimulus that arise from action on the target itself. In thecontext of this invention, these effects are manifest as phenotypicchanges. Of course, other effects, unrelated to the effect of thestimulus on the target may also elicit phenotypic changes. Thephenotypic differences between control and test cells are deemed to be“on-target” effects when these differences pertain to the presence orfunction of the target. For example, if a compound is being investigatedfor its ability to arrest mitosis, on-target effects include phenotypicmanifestations of arrested mitosis. If an image analysis shows that anunnaturally high percentage of cells exposed to the particular drug havetheir nuclear DNA in the mitotic state, as opposed to an interphasestate, then the drug may be deemed to have “on target” effects.

On the other hand, observed phenotypic differences between test andcontrol cells are deemed “off-target” effects when they do not pertainto the presence or function of the target. For example, in the arrestedmitosis target example, an observed deviation from control in thecompactness of the cells' Golgi apparatus may be deemed an off-targeteffect.

Because off-target effects are not apparently tied to the desired effecton a target, they may be suspect. In a sense, they represent“side-effects.” In a conservative view of drug discovery or otherbiochemical research endeavor, off-target effects may be viewed asindicia of toxicity. Of course, not all “side-effects” represent a toxicresponse. But in some cases, ideal drugs have only on-target effects.The use and relevance of off-target effects in biological investigationsis described more fully in U.S. patent application Ser. No. 10/621,821,filed Jul. 16, 2003, naming Kutsyy as inventors, and titled “Methods andApparatus for Investigating Side Effects,” which is incorporated hereinby reference for all purposes.

As explained elsewhere herein, indicia of hepatotoxicity or specifichepatic pathologies are provided by analyzing images of markers for thespecific pathologies. Accordingly, the image analysis methods andapparatus of the present invention may classify stimuli according to adegree of hepatotoxicity and/or a particular pathology. As explained,drug-induced hepatotoxicity in humans or animals can be classifiedaccording to specific cellular morphologies of hepatic injury.

Most agents that are acutely toxic to the liver lead to cytotoxic orcholestatic injury. Necrosis and apoptosis are forms of suchcytotoxicity. These conditions are manifest by specific and consistentphenotypic changes that can be detected by image analysis. Steatosis isanother form of cytotoxicity. It is characterized by an accumulation oflipid containing vacuoles within hepatocytes, which can be detected byimage analysis. Cholestasis is characterized by arrested bile flow andaccumulation of bile pigment within the hepatic parenchyma. Aspects ofcholestasis, such as modification of tight junctions between adjacentcells, can be detected by image analysis. Chronic injury caused bydrug-induced hepatotoxicity can lead to hepatitis, fibrosis, andcirrhosis. Image-based methods of this invention can identify some orall of these liver conditions.

With reference to FIG. 1A, shown is a cartoon representation of a groupof normal hepatocytes with various features as they might appear in theabsence of a toxic response. The group of hepatocytes 10 includesindividual hepatocytes 12 adjacent to each other, such that theindividual hepatocytes contact each other to form roughly block shapedbodies (as opposed to smooth roughly circular shaped bodies). At theinterfaces of individual hepatocytes 12, various tight junctions 20 canbe present. These tight junctions provide pathways for inter-cellulartransport between adjacent hepatocytes. It is believed that modificationof tight junctions may implicated in cholestasis and possibly othertoxic responses of hepatocytes.

A single hepatocyte cell 12 includes a nucleus 14, tubulin filaments 16,vacuoles 18, and various other organelles and features not shown in thepresent cartoon representation. The tubulin 16 is generally arranged asfibers that radiate from the nucleus 14 toward the periphery of the cell12. The vacuoles 18 are small lipid-containing regions dispersedthroughout the cytoplasm of cell 12. In addition, the cell includesactin filaments 22 concentrated at the edge of the cell appearing asdense actin cables that form a connected mesh works in the interior ofcell. Note that one of the hepatocytes is shown with two nuclei. It hasbeen found that as many as 30% or more of the healthy hepatocytes in agiven culture may have two nuclei (also designated as binuleate cells).

In a process 100 depicted in FIG. 1B, hepatocytes exposed to aparticular stimulus are imaged and then characterized by image analysisto provide information about hepatotoxicity. Understand that the processflow depicted in FIG. 1B is but one of many possible ways to implementthe present invention. Other approaches will classify hepatocytes usingdifferent pathologies and/or different orders of operations toaccomplish the same general result.

As depicted, the process 100 begins at block 101 where a culture ofprimary hepatocytes is obtained. These hepatocytes can be derived fromrats, humans, or other species, and should give consistent responseswhen exposed to a given stimulus. The hepatocytes may be cultured inmany different ways. Importantly, they should be cultured and presentedin manner that illuminates their susceptibility to the particularstimulus under investigation and allows the salient features of theirresponse to the captured in one or more images. Further discussion ofthe selection and culturing of primary hepatocytes for a hepatotoxicityassay and the support structures used to culture the primary hepatocytesis presented below in Section II: Culturing Hepatocytes for Assay.

Next, the process continues at a block 103 where the culturedhepatocytes are exposed to the stimulus under investigation. Asexplained above, many different stimuli can be tested in accordance withthis invention. In a typical example, hepatocytes in multiple wells arecontacted with a test compound. The compound may be provided atdifferent concentrations in different wells. In at least one well, acontrol well, no compound is used. In another case, the multiple wellsmay be contacted with the compound for different lengths of time priorto fixing. These approaches allow a generation of a stimulus responsepath. Each concentration or time point provides a different phenotypicpoint on the response path.

The next block in the depicted process (block 105) involves obtaining animage of the hepatocytes exposed to the stimulus under investigation.Single or multiple images are acquired from the same well. Frequently,multiple images will be taken at different “channels.” Each such channelmay be associated with a different marker, which highlights a specificfeature of the cell such as a particular macromolecule or organelle.Each separate image provides a different piece of information that canbe used to characterize the hepatocytes in culture.

In many embodiments, the individual cells of the image are separatelyanalyzed, apart from the background in the image, to obtain statisticalresults that characterize the impact of the stimulus on various featuresof the cells. To this end, the image must be “segmented” to separateregions of the image associated with individual cells. Each “segment” ofthe image is a group of contiguous pixels associated with an individualcell. Block 107 in flow chart 101 indicates segmentation.

With the image segmented, image analysis logic can evaluate those pixelsassociated with various individual hepatocytes in order to extractparticular features associated with an individual cell. In addition, thelogic classifies individual hepatocytes by toxic effect, includingindividual pathologies.

In flow chart 100, the separate consideration of individual cells isdepicted as a loop bracketed by blocks 109 through 113. In block 109,the process considers the “next” hepatocyte in the image. Note that,depending upon the processing capabilities of the image analysis system,multiple hepatocytes from the segmented image may be analyzed inparallel. Nevertheless, the analysis of each hepatocyte is a logicallydiscrete operation; hence the loop in process 100.

Once a hepatocyte is chosen at block 109, then the chosen hepatocyte isanalyzed and features are extracted from the image of the hepatocyte atblock 111. For instance, specific cell features, which signal particularmorphological, compositional, etc. aspects of the cell, can beextracted. These features may be associated with various cell componentsfor which markers are provided. Examples of such components includeGolgi, tubulin, actin, nuclear DNA, lipids, tight junctions, histones,activated caspase-3, and the like. Once features are extracted from thehepatocyte under consideration, then at 113, it is determined whetheradditional hepatocyte images remain to be analyzed. If so, then theprocess continues at 109. If not, then the process continues at block115.

Once the relevant features are extracted from the individual hepatocyteimages, then, at a block 115, the process derives a statistical resultfrom among the cells in the image for each relevant feature in thehepatotoxicity study. The statistical result is used to characterize theimpact of the stimulus on the relevant feature. The statistical resultcan be a mean value of a feature in question, an average value, astandard deviation, a variance, etc.

With the statistical results obtained for the set of hepatocytes, thedepicted process next determines whether the hepatocytes exhibitnecrosis (i.e., the stimulus induced necrosis). See decision 117. Next,the process may further classify the stimulus based upon whetherapoptosis occurred. See block 119. In each case, the cells have died,possibly as a result of exposure to the stimulus under consideration.

Next, the statistical result is used to determine if the stimulus shouldbe classified as inducing steatosis. See block 121. As explainedelsewhere herein, steatosis is a pathology manifest by the accumulationof lipid-containing vacuoles within the hepatocyte cytoplasm.

Next, the algorithm classifies the stimulus under consideration forcholestasis induction. As explained, cholestasis is characterized byarrested bile flow and accumulation of bile pigment within thehepatocyte. See block 123.

In alternative embodiments, alternative and/or additionalclassifications and analysis may be performed on the cell image underconsideration. Some of these will be described elsewhere herein. Otherswill be apparent to those of skill in the art. Note that the order ofthe pathology analysis presented above is not important to theinvention. The four pathologies can be considered in any order. Otherpathologies can be considered before or after those depicted in thefigure, or they may be interleaved with the pathologies in question.

When the cells have been classified according to pathology, the processmay be considered complete. However, it is frequently desirable toprovide image analysis information to a database that identifies thestimulus under consideration together with the image analysisinformation for individual hepatocytes and/or population statisticsassociated with all hepatocytes appearing in the image. See block 125.

Some of the terms used herein are not commonly used in the art. Otherterms may have multiple meanings in the art. Therefore, the followingdefinitions are provided as an aid to understanding the descriptionherein. The invention as set forth in the claims should not necessarilybe limited by these definitions.

The term “component” or “component of a cell” refers to a part of a cellhaving some interesting property that can be characterized by imageanalysis to derive biologically relevant information. General examplesof cell components include biomolecules and subcellular organelles.Specific examples of biomolecules that could serve as cell componentsinclude proteins and peptides, lipids, polysaccharides, nucleic acids,etc. Sometimes, the relevant component will include a group ofstructurally or functionally related biomolecules. Alternatively, thecomponent may represent a portion of a biomolecule such as apolysaccharide group on a protein, or a particular subsequence of anucleic acid or protein. Collections of molecules such as micells canalso serve as cellular components for use with this invention. Andsubcellular structures such as vesicles and organelles may also servethe purpose.

The term “marker” or “labeling agent” refers to materials thatspecifically bind to and label cell components. These markers orlabeling agents should be detectable in an image of the relevant cells.Typically, a labeling agent emits a signal whose intensity is related tothe concentration of the cell component to which the agent binds.Preferably, the signal intensity is directly proportional to theconcentration of the underlying cell component. The location of thesignal source (i.e., the position of the marker) should be detectable inan image of the relevant cells.

Preferably, the chosen marker binds specifically with its correspondingcellular component, regardless of location within the cell. Although inother embodiments, the chosen marker may bind to specific subsets of thecomponent of interest (e.g., it binds only to sequences of DNA orregions of a chromosome). The marker should provide a strong contrast toother features in a given image. To this end, the marker should beluminescent, radioactive, fluorescent, etc. Various stains and compoundsmay serve this purpose. Examples of such compounds include fluorescentlylabeled antibodies to the cellular component of interest, fluorescentintercalators, and fluorescent lectins. The antibodies may befluorescently labeled either directly or indirectly.

The term “stimulus” refers to something that may influence thebiological condition of a cell. Often the term will be synonymous with“agent” or “manipulation.” Stimuli may be materials, radiation(including all manner of electromagnetic and particle radiation), forces(including mechanical (e.g., gravitational), electrical, magnetic, andnuclear), fields, thermal energy, and the like. General examples ofmaterials that may be used as stimuli include organic and inorganicchemical compounds, biological materials such as nucleic acids,carbohydrates, proteins and peptides, lipids, various infectious agents,mixtures of the foregoing, and the like. Other general examples ofstimuli include non-ambient temperature, non-ambient pressure, acousticenergy, electromagnetic radiation of all frequencies, the lack of aparticular material (e.g., the lack of oxygen as in ischemia), temporalfactors, etc.

A particularly important class of stimuli is chemical compounds,including compounds that are drugs or drug candidates and compounds thatare present in the environment. The biological impact, includingtoxicity, of chemical compounds is frequently manifest as clearphenotypic changes.

Specific examples of biological stimuli include exposure to drugcandidate compounds, hormones, growth factors, antibodies, orextracellular matrix components. Or exposure to biologics such asinfective materials such as viruses that may be naturally occurringviruses or viruses engineered to express exogenous genes at variouslevels. Biological stimuli could also include delivery of antisensepolynucleotides by means such as gene transfection.

Other specific stimuli include exposure of cells to conditions thatpromote cell fusion. Specific physical stimuli could include exposingcells to shear stress under different rates of fluid flow, exposure ofcells to different temperatures, exposure of cells to vacuum or positivepressure, or exposure of cells to sonication. Another stimulus includesapplying centrifugal force. Other specific stimuli include changes ingravitational force, including sub-gravitation, application of aconstant or pulsed electrical current. Still other stimuli includephotobleaching, which in some embodiments may include prior addition ofa substance that would specifically mark areas to be photobleached bysubsequent light exposure. In addition, these types of stimuli may bevaried as to time of exposure, or cells could be subjected to multiplestimuli in various combinations and orders of addition. Of course, thetype of manipulation used depends upon the research endeavor at hand.

The term “phenotype” generally refers to the total appearance of anorganism or cell from an organism. In the context of this invention,cellular phenotypes and their representations in processing systems(e.g., computers) are particularly interesting. The phenotypiccharacteristics of a cell are functions of the cell's geneticconstitution and environment. Often a particular phenotype can becorrelated or associated with a particular biological condition ormechanism of action resulting from exposure to a stimulus. Generally,cells undergoing a change in biological conditions will undergo acorresponding change in phenotype. Thus, cellular phenotypic data andcharacterizations may be exploited to deduce mechanisms of action andother aspects of cellular responses to various stimuli.

A selected collection of data and characterizations that represent aphenotype of a given cell or group of cells is sometimes referred to asa “quantitative cellular phenotype.” This combination is also sometimesreferred to as a phenotypic fingerprint or just “fingerprint.” Themultiple cellular attributes or features of the quantitative phenotypecan be collectively stored and/or indexed, numerically or otherwise. Theattributes are typically quantified in the context of specific cellularcomponents or markers. Measured attributes useful for characterizing anassociated phenotype include morphological descriptors (e.g., size,shape, and/or location of the organelle) and composition (e.g.,concentration distribution of particular biomolecules within theorganelle). Other attributes include changes in a migration pattern, agrowth rate, cord formation, an extracellular matrix deposition, andeven cell count. Often, the attributes represent the collective value ofa feature over some or all cells in an image (e.g., some or all cells ina specific well of a plate). The collective value may be an average overall cells, a mean value, a maximum value, a minimum value or some otherstatistical representation of the values.

The quantitative phenotypes may themselves serve as individual points on“response curves.” A phenotypic response to stimulus may be determinedby exposing various cell lines to a stimulus of interest at variouslevels (e.g., doses of radiation or concentrations of a compound). Ineach level within this range, the phenotypic descriptors of interest aremeasured to generate quantitative phenotypes associated with levels ofstimulus.

The term “path” or “response curve” refers to the characterization of astimulus at various levels. For example, the path may characterize theeffect of a chemical applied at various concentrations or the effect ofelectromagnetic radiation provided to cells at various levels ofintensity or the effect of depriving a cell of various levels of anutrient. Mathematically, the path is made up of multiple points, eachat a different level of the stimulus. In accordance with this invention,each of these points (sometimes called signatures) is preferably acollection of parameters or characterizations describing some aspect ofa cell or collection of cells. Typically, at least some of theseparameters and/or characterizations are derived from images of thecells. In this regard, they represent quantitative phenotypes of thecells. In the sense that each point or signature in the path may containmore than one piece of information about a cell, the points may beviewed as arrays, vectors, matrices, etc. To the extent that the pathconnects points containing phenotypic information (separate quantitativephenotypes), the path itself may be viewed as a“concentration-independent phenotype.” The generation and use ofstimulus response paths are described in more detail in U.S. patentapplication Ser. No. 09/789,595, filed Feb. 20, 2001 naming Vaisberg etal., and titled, “CHARACTERIZING BIOLOGICAL STIMULI BY RESPONSE CURVES,”and U.S. patent application Ser. No. 10/623,485, filed on Jul. 18, 2003,naming V. Kutsyy, D. Coleman, and E. Vaisberg as inventors, and titled,“Characterizing Biological Stimuli by Response Curves,” both of whichare incorporated herein by reference for all purposes.

As used herein, the term “feature” refers to a phenotypic property of acell or population of cells. Typically, the points in a response curveof this invention are each comprised of multiple features. The terms“descriptor” and “attribute” may be used synonymously with “feature.”Features derived from cell images include both the basic “features”extracted from a cell image and the “biological characterizations”(including biological classifications such as cell cycle states). Thelatter example of a feature is typically obtained from an algorithm thatacts on a more basic feature. The basic features are typicallymorphological, concentration, and/or statistical values obtained byanalyzing a cell image showing the positions and concentrations of oneor more markers bound within the cells.

II. Culturing Hepatocytes for Assay

Hepatocyte cultures are used in assays designed to assesshepatotoxicity. Some are used as controls and others are exposed to oneor more stimuli that may produce toxic responses in hepatocytes. Asexplained, the cultures are imaged and analyzed to identify featuresthat may be affected by the stimuli tested. The features are analyzed inorder to categorize the stimulus according to pathology (or simplytoxicity), as will be described in more detail below.

Hepatocyte cultures may be derived from rats, humans or other speciesappropriate to the stimulus under investigation. Generally, hepatocytesused in different experiments should give consistent responses whenexposed to the same assay conditions. Preferably, therefore, they comefrom a relatively homogeneous pool so that cells taken from one sourcerespond similarly to cells taken from a different source. Laboratoryrats, being relatively homogeneous genetically in comparison to mosthuman groups, may provide a suitably consistent source of hepatocytesfor assays of this invention. However, the effects of a stimulus on rathepatocytes sometimes fail to adequately represent the effects seen onhuman hepatocytes. Hence, human hepatocytes may be necessary for someinvestigations.

Transformed or immortalized human hepatocyte cell lines can provide agenetically homogeneous source for many assays. One widely usedtransformed hepatocyte cell line is HepG2 available from the AmericanType Culture Center as HB 8065. Hep G2 is also available from AmphioxusCell Technologies of Houston, Tex.

Unfortunately, immortalized cells do not always provide a completelyrealistic model of normal hepatocytes. In particular, although thehepatoma derived cell lines are easy to culture and maintain, they maynot express the full complement of Cytochrome P450 metabolizing enzymes.One approach to this problem is to genetically modify the immortalizedcells to mimic the expression pattern of a non-immortalized cell.

For non-immortalized or primary cells, one may use eitherfreshly-isolated cells, which have been recently harvested, orcryopreserved cells. Several commercial vendors provide fresh orcryopreserved primary hepatocytes from human, rat, dog, and primatespecies. These vendors include Xenotech LLC of Lenexa, Tex.; TissueTransformation Technologies of Edison, N.J.; In Vitro Technologies ofBaltimore, Md.; Gentest (a BD Biosciences company) of Woburn, Mass.; andBD Biosciences. Transplant ready/fresh human hepatocytes are availablefrom In Vitro Technologies and Tissue Transformation Technologies.Freshly isolated cells are normally used within 24 hrs after harvesting.For some experiments, it is possible to use them at 72 hrs afterharvest.

Although non-immortalized cells generally present a better model forhepatoxicity in assays than their immortalized counterparts,immortalized cells are usually easier to use. As indicated, primaryhuman hepatocytes have a finite shelf-life and exhibit significantgenetic variation across samples. Accordingly, another approach toculturing hepatocytes for an assay can include isolating a highlydifferentiated hepatocyte cell line that retains the metabolic activityof primary hepatocytes but has been immortalized to allow easycultivation in vitro. For a more detailed description of establishedtechniques for preparing such cell lines, see Kobayashi, et al.,“Prevention of acute liver failure in rats with reversibly immortalizedhuman hepatocytes,” Science, 287:1258-1262 (2000).

Hepatocyte cultures can be grown in or on various support structures.For instance, a bare plastic support that includes nutrients can be usedto support a culture. Similarly, a glass surface can be used to supporta culture. Other kinds of supports can include extra-cellular matricessuch as collagen or Matrigel (available from BD Biosciences, San Jose,Calif.). Such structures can be provided in multiwell plates, such as384-well assay plates. Cultures of primary hepatocytes generally growwell in three-dimensional lattice structures.

In some embodiments, hepatocytes can be cultured with associated cellsto encourage the hepatocytes to behave naturally in an assay. Forinstance, hepatocytes can be co-cultured with stromal cells such asfibroblasts. Co-culturing hepatocytes and support cells in this mannermay improve the predictive qualities of the assays in some contexts.

A discussion of co-culturing is provided in U.S. Pat. No. 6,599,694 ofElias (issued Jul. 29, 2003), which is incorporated herein by referencefor all purposes. As explained there, two separate cell types areexposed to a stimulus suspected of producing a biological condition(e.g., a specific toxic pathology). The two different cell types areco-cultured or otherwise allowed to interact with one another before andduring exposure to the agent. The images of the cells show how thestimulus separately affects each of the cell types. Specifically, theimages show how the phenotype of each cell type changes (or does notchange) upon exposure to the stimulus. In this regard, the concept of aphenotype encompasses visual indicators showing migration patterns,growth rates, extracellular matrix depositions, etc.

In the context of this invention, the cultures and supports describedabove are intended to provide in vitro models of in vivo hepatocytefunctioning. For example, a three-dimensional co-culture of primaryhuman liver stroma and parenchymal cells can be provided in vitro in amanner that mimics in vivo liver tissue function.

For some assays, such as the cholestasis assay, it may be appropriate toculture “polarized hepatocytes.” Such culture can mimic features of thebiliary tree, such as bile ducts, and thereby cause the hepatocytes tosecrete bile into a “duct” (the exposed portion of the culture) andotherwise behave as if they were part the biliary tree. Becausecholestasis is characterized by inhibition of bile flow, such culturingfacilitates characterization of cholestasis.

An exemplary procedure for preparing hepatocyte cultures for use withassays of this invention will now be described. Specifically, theprocedure involves the use of primary rat hepatocytes as follows:

Isolation and Culture of Primary Rat Hepatocytes

-   1. Adult Sprague-Dawley male rats (250-350 g) are anesthetized with    Isoflurane to induce an anesthetic plane by inhalation.-   2. An initial incision is made with surgical scissors to the    ribcage, proximal to the pubis through the skin and muscle wall,    with care taken to avoid cutting the diaphragm.-   3. The intestines are then moved from the abdominal cavity to the    animal's side. The portal vein and inferior vena cava (IVC) are    exposed and two surgical sutures are loosely placed around the    portal vein.-   4. The IVC is injected with 0.3 cc heparin (1000 U/mL). The    peristaltic pump is turned on and the flow rate is set to 1 mL/min    using Liver Perfusion Medium warmed to 37° C. (available from Gibco    BRL, Div. of Life Technologies Inc., Gaithersburg, Md., Catalog    #17701).-   5. The portal vein is cannulated and the portal vein sutures are    tightened. With care taken to avoid introducing any air bubbles, the    perfusion line is connected to the catheter.-   6. The animal is euthanized by cutting the heart and diaphragm. The    rate of the peristaltic pump is slowly brought to 16 mL/min and    perfused for 15 minutes.-   7. After 15 minutes, the perfusion media is switched to Liver Digest    Medium (Gibco BRL, Div. of Life Technologies Inc., Gaithersburg,    Md., Catalog #17703) at 37° C. and perfused for 15 minutes.-   8. The liver is removed and placed in a 10 cm dish. Next, the liver    is minced with small scissors and the contents are poured into a    sterile flask with 50 mL of DMEM supplemented with 100 U Dnase I.-   9. The flask is incubated at 37° C. for 20 minutes. The cells are    strained through sterile 100 micron mesh nylon.-   10. The contents are centrifuged for 5 minutes at 50 g. The    supernatant is then removed and the pellet is resuspended in 10 mL    DMEM.-   11. The hepatocytes are purified in 30% Percoll and centrifuged at    50× g for 5 minutes. The supernatant is discarded and the pellet is    resuspended in 5 mL DMEM medium.-   12. The cells are counted by Trypan blue exclusion.-   13. Twenty five to fifty thousand cells/well are plated in a 96 flat    well plate pre-coated with collagen I (BD Biosciences, San Jose,    Calif. or CellzDirect, Tucson, Ariz.) or custom coated with 300    micrograms/ml collagen I in PBS (phosphate buffered saline). The    plates are incubated at 37° C./5% CO₂ in HCM (Cambrex Bioscience,    Baltimore, Md.), Hepatozyme (Invitrogen, Carlsbad, Calif.) or Cell    Plating Medium (CellzDirect, Tucson, Ariz.) for two hours. Cells are    washed, and the medium is replaced with Hepatozyme containing 1-30    micrograms/ml of Matrigel (BD Biosciences) or collagen I, and the    plates are incubated overnight.

As appreciated by those of skill in the art, other procedures forpreparing hepatocyte cultures can also be used within the scope of thepresent invention.

III. Imaging and Segmentation

As indicated, the phenotypic data characterizing stimuli is derived, atleast in part, from images of hepatocytes exposed to particularcombinations of stimulus type and stimulus level. See block 105 in FIG.1B, for example. Various techniques for preparing and imagingappropriately treated cells are described in the following U.S. patentapplication Ser. No. 09/310,879 by Crompton et al., entitled A DATABASEMETHOD FOR PREDICITIVE CELLULAR BIONINFORMATICS, filed on May 14, 1999;U.S. Pat. No. 6,651,008 by Crompton et al., issued Nov. 18, 2003; andU.S. Pat. No. 6,631,331 by Crompton et al., issued Oct. 7, 2003, each ofwhich are incorporated by reference herein for all purposes.

Generally the images used as the starting point for the methods of thisinvention are obtained from cells that have been specially treatedand/or imaged under conditions that contrast the cell's markedcomponents with other cellular components and the background of theimage. Typically, the cells are fixed, optionally washed, and thentreated with a material that binds to the components of interest andshows up in an image (i.e., the marker). Preferably, the chosen agentspecifically binds to the cellular component of interest, but not tomost other cellular biomolecules. In some cases, the cells are treatedwith the marker prior to fixation. This may be appropriate when, forexample, the marker is used to distinguish live and dead cells.

In the case of cells treated with a fluorescent marker, a collection ofsuch cells is illuminated with light at an excitation frequency. Adetector is tuned to collect light at an emission frequency. Thecollected light is used to generate an image, which highlights regionsof high marker concentration.

Additional operations may be performed prior to, during, or after theimaging operation (105) of FIG. 1B. For example, “quality controlalgorithms” may be employed to discard image data based on, for example,poor exposure, focus failures, foreign objects, and other imagingfailures. Generally, problem images can be identified by abnormalintensities and/or spatial statistics.

In a specific embodiment, a correction algorithm may be applied prior tosegmentation to correct for changing light conditions, positions ofwells, etc. In one example, a noise reduction technique such as medianfiltering is employed. Then a correction for spatial differences inintensity may be employed. In one example, the spatial correctioncomprises a separate model for each image (or group of images). Thesemodels may be generated by separately summing or averaging all pixelvalues in the x-direction for each value of y and then separatelysumming or averaging all pixel values in the y direction for each valueof x. In this manner, a parabolic set of correction values is generatedfor the image or images under consideration. Applying the correctionvalues to the image adjusts for optical system non-linearities,mis-positioning of wells during imaging, etc.

Note that the quality of the images is dependent on cell plating,compound dilution, compound addition and imaging focusing. Failures inany these systems can be detected by a variety of methods. For example,cell plating could fail because of a clogged tip in a delivery pipette.Such failure can be identified by adding a fluorescent dye or bead tothe cell suspension. The fluorescence of this dye or bead is chosen tobe at a different channel (wavelength) than the markers used to imagecellular components. Another potential failure could occur duringcompound delivery. To detect such failures, one can add a fluorescentdye or bead in the compound plate before compound dilution. The amountof fluorescent dye or bead is proportional to the amount of compound.Yet another potential problem occurs when the focus of the imageacquisition system changes during imaging. To account for such spatialbiases, one can employ control wells containing, for example, cells withno or neutral compounds interspersed throughout the plate. Still anotherproblem results from foreign objects (e.g., small dust particles) in thewell. This can be addressed with image segmentation and statisticaloutlier identification techniques. Both manual and automated methods canbe used to eliminate bad images from analysis.

In order for the images to provide useful information about individualhepatocytes, the hepatocytes should be well spaced and distinguishablefrom one another. Otherwise, segmentation may be difficult orimpossible. If cell clumping makes identification of individualhepatocytes difficult, the cells can be seeded at a lower density ordifferent culturing conditions can be used.

Growing cells on a three-dimensional matrix such as collagen or Matrigelmay also present some challenges for imaging. In particular,autofocusing can be difficult when cells are located in athree-dimensional structure. However, culturing conditions and automatedmicroscopy capabilities can be adjusted to keep a sufficient number ofcells within an accessible focal plane. Furthermore, the spatialresolution can be adjusted according to the degree of magnificationnecessary for a particular assay. Other conditions that can be modifiedaccording to the segmentation needs of a particular assay include theuse of markers (e.g., DAPI for DNA) and the cell fixation proceduresimplemented. An example of an automated microscopy system suitable foruse with this invention is the Image Express available from AxonInstruments or the Discovery 1 available from Universal Imaging andMolecular Devices.

The goal of segmentation is to allow feature extraction on acell-by-cell basis. Segmentation identifies discrete regions of an imagethat include only those pixels where the components of a single cell aredeemed to be present. Thus, each representation is a bounded collectionof pixels, each providing associated features characterizing a singlecell.

Segmentation can be accomplished in numerous ways. These include use oftechniques that identify regions of high DNA concentration (presumed tobe nuclear regions) and watershed algorithms. Typically nuclear DNAmarkers provide a strong signal and there is a high contrast in theimage and an edge detection based segmentation process can be used. Thesegmentation process typically identifies edges where there is a suddenchange in intensity of the cells in the image and then looks for closedconnected edges in order to identify an object. In some cases,segmentation can be conducted on confluent or semi-confluent cultures

In one approach to segmentation, the image analysis tool initiallyidentifies the nucleus of each cell captured in the image underconsideration. If images from different channels are well registered,the nuclei can be first identified in the DNA channel and then overlaidto the image under consideration. The segmentation algorithm defines a“ring region” around each nucleus. Generally, this step serves to definethe perinuclear region. This region encompasses some or all of thecytoplasmic cell components in a normal interphase cell. The generalmethod is described in U.S. Published Patent Application No.US-2002-0141631-A1, published Oct. 3, 2002, naming Vaisberg, Cong, andWu as inventors, and titled “IMAGE ANALYSIS OF THE GOLGI COMPLEX,” whichis incorporated herein by reference for all purposes.

To identify bi-nuclear cells (and not treat each nuclei as the locus ofdifferent cell), one may employ nearest neighbor algorithms and otheralgorithms of the type described in U.S. patent application Ser. No.10/615,116, filed Jul. 7, 2003, naming Coleman et al., and titled“METHODS AND APPARATUS FOR CHARACTERISING CELLS AND TREATMENTS,” whichis incorporated herein by reference for all purposes.

A watershed algorithm has been found to provide very good segmentationeven in cultures containing many abutting hepatocytes or cells of othertypes. A suitable algorithm for this purpose is described in U.S.Published Patent Application No. US-2002-0154798-A1, published Oct. 24,2002, naming Cong and Vaisberg as inventors and, titled “EXTRACTINGSHAPE INFORMATION CONTAINED IN CELL IMAGES.” which is incorporatedherein by reference for all purposes. The watershed approach does aparticularly good job of correctly illustrating the shape of cellsidentified during segmentation. In some embodiments, employs image datafor a cell shape-indicative marker (for example, cytoskeletalcomponents, (e.g., tubulin), one or more cytoplasmic proteins (forexample lactate dehydrogenase or total cell protein), or membranecomponents (e.g., lipids or plasma membrane receptors)) in a watershedtechnique. Markers that detect proteins localized on the cell surfacemay work well in this technique. Examples include the tight junctionproteins zonula occludens-1 (ZO-1), ZO-2, and ZO-3, which are found atthe interface of fused hepatocytes and other cells. Other reagents forsegmenting cells include succinimidyl esters conjugated to fluorescentdyes such as TAMRA or Alexa (Molecular Probes, Eugene, Oreg.). Thisreagent labels the primary amine groups of proteins and can be used tolabel any cell, including hepatocytes.

The following example is a procedure for fixing and staining primary rathepatocytes to visualize nuclei, golgi, and actin. The Alexa 647 nmsuccinimidyl ester reagent (A647SE) is used to segment individualhepatocytes within an image.

-   1) To each well containing 100 ul of medium, add 250 μl of fix    solution (5.3% paraformaldehyde, in 1× PBS) and incubate at room    temperature for two hours.-   2) Remove the fix solution (by inverting the plate upside down), and    add 350 μl of wash buffer (1× PBS, 0.2% Triton X-100) to each well.    Incubate for 5 minutes at room temperature.-   3) Remove the wash buffer, and add 100 μl of block buffer (10% heat    inactivated normal goat serum (Vector Labs, Burlingame, Calif.    diluted in 1× PBS, 0.2% TritonX-100). Incubate for 30 min at room    temperature.-   4) Remove the block buffer and add 100 μl of staining mix per well.    Staining mix is comprised of Alexfluo488 conjugated phalloidin    (1:200 dilution of a 20 μM stock solution), and rhodamine conjugated    DM1a (1:300 dilution of 1 mg/ml stock solution) diluted in block    buffer. Incubate overnight in the dark at 4° C.-   5) Aspirate off the staining mix, and add 100 ul per well of A647SE    mix: 0.5 μg/ml A647SE (stock 10 mg/ml) and 1:1000 dilution of    Hoechst 33342 (stock 5 mg/ml) in wash buffer. Incubate at room    temperature for one hour in the dark.-   6) Aspirate off the staining mix, and add 350 μl wash buffer per    well. Incubate 5 min at room temperature.-   7) Repeat the wash step two times.-   8) Seal the plate and store at room temperature in the dark prior to    imaging.    IV. Detecting Necrosis and Apoptosis

Acute liver failure can be associated with such necrosis and apoptosis.Both of these pathologies are a manifestation of cell death. Yet, theyhave distinct features that can be utilized in image analysis tocharacterize a cell as necrotic, apoptotic, or neither apoptotic nornecrotic.

To determine whether hepatocytes are necrotic, various features can beassayed. General indicators of necrosis include increased membranepermeability, decreased enzyme activity, diffuse Golgi distribution ininterphase cells, dilated mitochondria morphology, and compromisedmetabolic activity of mitochondria.

In one embodiment, an assay tests the membrane permeability of thetreated hepatocytes. Generally, cells with highly permeable membranesare deemed to be necrotic. Accordingly, a stain that is not permeable tothe cell membrane of live cells can be introduced to a culture oftreated hepatocytes before the hepatocytes are fixed for imaging. Oneexample of a stain that can be used is ethidium bromide homodimer(available from Molecular Probes, Inc., Eugene, Oreg.). The materialstrongly binds to nucleic acids, yet is substantially impermeable to thecell membranes of live cells. It has been found ethidium bromidehomodimer binds to the DNA of dead cells with an approximately 40-foldincrease in fluorescence intensity. Other impermeable DNA stains (e.g.,acridine orange, propidium ionide) or other impermeable cellular stains(e.g., trypan blue or rhodmine labeled phalloidin, which labels actinfilaments) can also be used.

Examples of cell enzymes that can be used to generally identify necroticcells include esterases. Biochemical assays include measuring ATPlevels, or the reduction of a tetrazolium salt into a colored formazonproduct (MTS or MTT assay). In one embodiment, a live cell stain such ascalcein AM (available from Molecular Probes, Inc., Eugene, Oreg.) can beintroduced to a treated hepatocyte culture prior to fixing. Calcein AMis a non-fluorescent dye that is cell permeable. The dye becomesfluorescent after cleavage of its ester groups by cellular esterases.This requires cellular energy, and is therefore a good indicator of livecells having intact membranes and functional mitrochondria with intactATP production machinery. The dye can also be used to observe live cellmorphologies such as membrane blebbing, which is an early indicator ofapoptosis. Although calcein AM is bright in live cells, its fluorescenceintensity decreases with time after the cells are fixed. Hence, in someembodiments, the hepatocytes may be imaged prior to fixing. In otherembodiments, the cells are imaged relatively soon after fixing. Ineither case, strongly fluorescing cells are likely to be live andnon-fluorescing cells are likely to be necrotic. Another example of astain that can be used either before or after fixing is CMFDA (i.e.,5-chloromethylfluorescein diacetate), which provides a good indicator oflive cells. Like calcein AM, it penetrates the cell membrane. In livingcells with esterase activity, its acetate group is cleaved to produce agreen fluorescent protein (excitation 485 nm, emission 520 nm), which istrapped inside the live cell.

Diffuse Golgi can be identified by imaging cells stained with a Golgibinding marker such as labeled Lens culinaris lectin (LC lectin) orantibodies to proteins enriched in the Golgi complex, such as gp130,[beta]COP. From imaged cells, the Golgi can be characterized by locatingthe region in a cell where the Golgi resides, and mathematicallyanalyzing the region for peakedness, texture, amount of Golgi complex inthe region, etc. As specific examples, the mathematical characterizationof the Golgi complex include the kurtosis of intensity values obtainedfrom the image, eigenvalues of a singular value decomposition ofintensity values obtained from the image, and at least one of a mean anda standard deviation of intensity values obtained from the image. Asuitable procedure for characterizing Golgi is described in U.S.Published Patent Application No. US-2002-0141631-A1, published Oct. 3,2002, which was previously incorporated herein by reference.

In addition to determining whether cells are necrotic, a determinationcan be made about whether the cells are apoptotic. Apoptosis is a formof cell death and follows a specific biochemical cascade of events.Generally, apoptosis may be activated in cells by an intrinsic programof gene activations or by exposure to harmful stimuli. Apoptosis ischaracterized by a pathway that includes changes in certain membraneproteins, depolarization of the mitochondrial membrane, release ofcytochrome C from mitochondria, activation of various caspase enzymes(caspase 3 is a major isoform involved in apoptosis), condensation,fragmentation and granularization of the nuclei, and breakdown ofvarious nuclear and cellular proteins including actin, and microtubules.In addition, apoptotic cells tend to become loosely attached to theirsubstrate and can removed easily. Various of these specificmanifestations of apoptosis that can be identified by image analysisinclude exposure of phosphatidyl serines on membrane proteins, themigration of cytochrome c from the mitrochondria into other regions ofthe cell, changes of mitochondrial membrane potential, activation ofcaspase 3, cleavage of caspase substrates (PARP, microtubule and actin),and condensation, fragmentation and granularization of the nuclei.

FIG. 2 presents a cartoon depiction of a hepatocyte undergoing apoptosisin a hepatocyte 212. In comparison to a normal hepatocyte 12, theapoptotic cell has become smaller and more rounded. Furthermore, itsnucleus 216 has condensed and become fragmented. Thus, markers for itsnuclear components may exhibit brighter than normal signals.Furthermore, the tubulin fibers 218 appear less discrete as fiberbundles and more diffusely distributed throughout the cytoplasm. Incontrast, the tubulin fibers 18 in a normal hepatocyte are disposed asthick tubulin bundles radially with respect to the nucleus 16 as shown.

Any one or more of the apoptosis features depicted in FIG. 2 ordescribed elsewhere here may be employed to identify apoptotic cells inan image analysis procedure of this invention. A specific image-analysisalgorithm for identifying necrotic and apoptotic hepatocytes isdescribed below in the context of FIG. 3.

In one embodiment, annexin V is used to detect whether a cell isapoptotic. Annexin V is a commercially available marker from R&D Systemsof Minneapolis, Minn. Annexin V is a member of a calcium andphospholipid binding family of proteins with vascular anticoagulantactivity. Various synomyms for annexin V exist: placental protein 4(PP4), placental anticoagulant protein I (PAP I), calphobindin I(CPB-I), calcium dependent phospholipid binding protein 33 (CaBP33),vascular anticoagulant protein alpha (VACa), anchorin CII, lipocortin-V,endonexin II, and thromboplastin inhibitor. Largely found on thecytosolic face of plasma membranes, this molecule has high affinity forphospholipids in the presence of physiological concentrations ofcalcium. See, Cookson, B. T. et al. (1994) Genomics 20:463; Grundmann,U. et al. (1988) Proc. Natl. Acad. Sci. USA 85:3708; and Huber, R. etal. (1992) J. Mol. Biol. 223:683, each of which is incorporated hereinby reference.

During apoptosis, the proteins reverse orientation within the membraneto expose the phosphatidyl serine residues. Annexin V binds to theprotein in this state. However, Annexin V does not bind to the proteinwhen it is in its normal orientation. Accordingly, when annexin Vbinding is detected, the cell's death might be attributed to apoptosis.For purposes of an image analysis assay, a fluorescently labeled annexinV may be used.

In another approach, late-stage apoptosis can be detected by using dyesthat are sensitive to changes in mitochondrial membrane potential, suchas MitoTracker (available from Molecular Probes, Inc., Eugene, Oreg.).Normally, the mitochondrial membranes have a strong electrochemicalgradient that drives ATP synthesis. During apoptosis, the mitochondriawithin a cell depolarize. Loss of mitochondrial membrane potential hasbeen recognized as an early marker of apoptosis and is quantifiable byMitoTracker® Red

In other embodiments, detection of cytochrome C outside of themitochondria can be an indicator of apoptosis. Cytochrome C is requiredfor respiration in the mitochondria, and detection of cytochrome Coutside of the mitochondria indicates that the mitochondria have beencompromised. More particularly, when the mitochondria stops functioning,the mitochondria releases cytochrome C. Green Fluorescent Protein (GFP)and/or antibodies also can be used to identify the presence ofcytochrome C outside the mitochondria. See, e.g., Goldstein et al.(2000) Nature Cell Biol. 2:156; and Ogawa et al. (2002) Intl. J.Molecular Medicine 10:263.

In another embodiment, early-stage apoptosis can be detected by thepresence of activated caspase 3, which is central to the apoptoticcascade. Different caspases may be activated, depending on the nature ofthe stimuli. Caspase 3, however, appears to be a key convergence pointof most apoptotic pathways and its activation is an important hallmarkof the apoptotic response. One method to detect caspase 3 activation isto use an antibody specific for activated caspase3 (SignalingTechnology, Inc., Beverly, Mass.) in an immunoprecipitation experiment.In addition, there are modified substrates for caspase 3 that can beused in biochemical assays such as Asp-Glu-Val-Asp-AMC available fromMolecular Probes, Inc., Eugene, Oreg.). Cleavage of the peptide bycaspase 3 yields the blue fluorescent AMC product (7-amino-4-methylcoumarin). The same peptide substrate can be conjugated to rhodamine 110(Molecular Probes) to yield a fluorescent product after cleavage bycaspase 3. Another example is a FITC conjugated caspase3 inhibitor(caspTag, Serologicals, Norcross, Calif.), which recognizes andcovalently link itself to the activated caspase3.

In yet another embodiment, specific labeling of fragmented DNA is usedto determine whether the cell died by apoptosis. During apoptosis,enzymatic cleavage of the nuclear DNA occurs. This can be observed by a“TUNEL” assay that measures terminal transferase activity, whichindicates the number of free ends of DNA generated by the degradation.An alternative to TUNEL is to use an antibody that recognizes HistoneH2A.X phosphorylation (Upstate Cell Signaling, Waltham, Mass.) inresponse to DNA damage and strand breakage that occurs during apoptosis.

In addition, the effect on the nucleus can be observed by imaging thenucleus of a cell. In particular, the cell can be treated with a DNAstain (e.g., fluorescently labeled antibodies to DNA and fluorescent DNAintercalators such DAPI and Hoechst 33341 available from MolecularProbes, Inc. of Eugene, Oreg.) and then imaged to assess the cell basedon the morphology of its nucleus. Apoptosis is often accompanied bycondensation and fragmentation of the nucleus. Therefore indicia ofapoptosis include relatively small nuclear regions of high signalintensity and “punctate” nuclear regions in which the nuclei separateinto small points.

Note that condensed punctate nuclear regions are also found in mitoticcells. Thus, methods for characterizing mitosis may be used—inconjunction with other methods specific for apoptosis—to characterizethe ability of a stimulus to induce apoptosis. “Mitotic index” is onemeasure of the mitotic state of cells. Generally, the mitotic indexrefers to the proportion of mitotic cells in the cell population. Thecell cycle state (including the mitotic state) can be determined byvarious techniques such as those described in U.S. patent applicationSer. No. 09/729,754, filed Dec. 4, 2000, naming Vaisberg et al., andtitled “CLASSIFYING CELLS BASED ON INFORMATION CONTAINED IN CELLIMAGES,” which is incorporated herein by reference for all purposes. Inone approach, the mitotic index is determined from the intensity of theDNA signal in cells (mitotic cells have twice as much DNA as G1 cells)and the variance in the DNA signal intensity (greater variance suggestsmitosis).

Another property of cells undergoing apoptosis is that they tend tobecome loosely attached to the substrate. Both cytoplasm shrinkage andloss of attachment is probably a result of cytoskeleton damage bycaspases. This property can be detected by exposing the culture to atreatment that will tend to dislodge and remove loosely attached cells.One way to accomplish this is by carefully washing a cell culture underconsideration. The level of apoptosis has been found to correlate wellto a “washout coefficient” based on cell counts in washed and unwashedcultures exposed to a stimulus suspected of inducing apoptosis; e.g.,(cc (unwashed)—cc(washed))/cc(unwashed).

It has been found that hepatocytes, cultured on_a collagen substratumsurface, stained using standard immunocytochemistry procedures, andwashed using phosphate-buffered saline or Tris-buffered saline givewashout coefficients that correlate strongly with apoptosis. Forexample, it has been found that approximately 40% or higher percentageof the cells exposed to a strong apoptosis inducing stimulus becomedislodged during washing. Generally, large values of the coefficientindicate that the associated stimulus is a strong inducer of apoptosis.

Aside from washing the culture, centrifugation, shear force, mixing, andthe like also can be used to dislodge apoptotic cells and provideclassifying information corresponding to washouts.

In some embodiments, apoptosis can be analyzed using time-lapse imagingtechnology. As described above, an apoptotic event is characterized bycondensation of the nuclei. A specific example of a time-lapse apoptosisexperiment will now be described. Using multi-site time-lapse imaging oflive cells expressing a GFP-histone2B (or any other GFP-tagged proteinthat functionally co-localizes with DNA) at low (5×-10×) magnification,the apoptotic event can be observed. Cells can be kept alive in theirpreferred environment using an environmental chamber with heat andcarbon dioxide. Many wells can be sequentially visited and images can betaken. This process can be repeated every 10-15 minutes over a course ofdays in the presence of a compound or control, until hundreds of imagesare collected that can be collated into movies and analyzedqualitatively or quantitatively. Three structural pathways that thecells can take to an apoptotic death include the following.

First, some of the more traditional apoptotic inducers (e.g.,Staurosporine, Tpen) cause cells to apoptose from interphase (G1, S, orG2). This can be visualized as condensation of the nuclei into a singlesmall and bright condensed fragment.

Second, compounds that cause mitotic arrest (e.g., Taxol) cause cells toapoptose from mitotic (or arrested mitosis as the case may be). Cellsthat apoptose from this state result in a large array of multiple smallDNA fragments. This may be a form of mitotic catastrophe.

Third, cells that are arrested in mitosis for a long period of time(e.g., with Taxol) may also decondense their DNA and become live,albeit, multinuclear cells. These cells will proceed to apoptose with alarge array of multiple small DNA fragments.

The apoptotic states induced by second and third pathways above aresensitive to perturbations in the liquid layer, such that the cells aredisturbed even in a “homogenous” assay by the addition of stain, withoutwashing. The apoptotic state induced by first pathway is also easilywashed away, but less easily than with the second and third pathways.

Using multi-site time-lapse techniques as described, apoptotic eventscan be quantified on a cell-by-cell basis over time to extract kineticinformation.

FIG. 3 presents a flow chart highlighting some operations that may beused in an assay for apoptosis and necrosis in accordance with anembodiment of this invention. A depicted process 301 begins after thehepatocytes in two or more samples (e.g., hepatocyte cultures inseparate wells of a plate) have been exposed to a stimulus as indicatedat block 103 of FIG. 1B. Generally, the exact same stimulus is providedto the two or more hepatocyte samples. Then each of them is subject to adifferent set of preparatory treatments.

Importantly, one sample is washed and the other is not. Optionally, thesamples are treated with different markers. As illustrated in a block303, one hepatocyte sample is treated in a single step that does notinclude a wash step. In one specific embodiment, this step involvescontacting the hepatocytes with a fixing agent, an esterase activitymarker such as calcein AM, a cell membrane impermeable marker such asethidium bromide homodimer, and a DNA intercalating stain such asHoechst.

A second sample is subjected to a multi-step preparatory treatment 305that includes one or more washes. One example of a multi-steppreparatory treatment is a quadstain. This allows comparison of washedand unwashed samples to assess the stimulus' impact on cell adhesion. Inone embodiment, the treatment includes contact with a tubulin marker(e.g., DM1-α), an actin marker (e.g, fluorescently labeled phalloidin),a DNA marker, a caspase-3 activity marker. In a specific example, thetreatment includes the following sequence:

Cell plates are split into two groups: a one-step group and a quad-staingroup. For the one step group, calceinAM and ethidium homodimer areadded to the wells and the plates incubated for 30 min at 37 degrees. Afixative (4% paraformaldehyde, 5 ug/ml Hoechst 33342 in phosphatebuffered saline) is added to each well and the plates are imaged. Thefollowing steps are used to treat wells in the quad-stain group (1)fixative, (2) wash buffer (e.g. phosphate buffered saline) (3) blockingbuffer (Technova, catalog #T1682) (4) antibody cocktail with markers(Hoechst 3341, caspase3 antibody, rhodamine labeled DM1-α, andfluorescently labeled phalloidin), (5) wash, (6) incubation with afluorescent secondary antibody to the caspase primary antibody, (7)wash, and (8) image. Hoechst 3341 is a DNA marker, caspase 3 is anapoptosis marker, DM1-α is a microtubule marker, and phalloidin is anactin marker.

After the hepatocyte samples have been separately treated as described(operations 303 and 305), they are imaged and segmented as indicated atblock 307. This corresponds to operations 105 and 107 of FIG. 1B. Next,the images are separately analyzed to extract pertinent features on acell-by-cell basis. See block 309. This corresponds generally tooperations 109, 111, and 113 of FIG. 1B. From this point, the flow chartbranches to analyze the images for apoptosis (one branch) and necrosis(a second branch). The analysis employs features extracted from bothsamples (the one receiving treatment 303 and the other receivingtreatment 305).

On the necrosis branch, the method identifies average levels of esteraseactivity (e.g., calcein AM activity) and cell permeability (e.g.,ethidium bromide homodimer binding) in cells of the image. See block311. This operation represents a specific version of the “derivestatistical result for each relevant feature” operation of FIG. 1B(block 115). From this information, the method characterizes thenecrotic response of the hepatocytes. See block 313. In a specificembodiment, necrotic cells have low esterase activity and high cellpermeability, while live cells have high esterase activity and low cellpermeability. These features are obtained as statistical parametersacross all cells in the image.

On the apoptosis branch, the method considers the condensation and/orfragmentation of the nuclei (using mitotic index for example), celladhesion (using a washout coefficient for example), and averagecaspase-3 activity (or the activity of some other apoptosis marker). Seeblock 315. As with operation 311 on the necrosis branch, operation 315represents a specific embodiment of operation 115 from FIG. 1B. Afterthe information relevant to apoptosis has been identified andconsidered, the method characterizes the apoptotic effect of thestimulus. See block 317. As discussed above, apoptosis is characterizedby a lowering of cell adhesion, condensation of the nuclei, andincreased caspase 3 activity—among other effects.

Note that the above assay method is but one of many apoptosis and/ornecrosis assays within the scope of this invention. Various combinationsof markers and features can be employed in image analysis techniques toassess the effects of stimuli on hepatocytes.

Another assay for apoptosis has been applied to cancer cells. And itshould be generally applicable to other cell types as well. In theassay, cells exposed to a stimulus are divided into two samples. Thefirst group is treated very simply; it is fixed and contacted with a DNAmarker in a single step. In other words, it is treated with a cocktailcontaining only a fixative agent and a DNA marker (e.g., the Hoechst33341 marker). Thereafter it is imaged without first washing. The secondsample is subjected to a more elaborate procedure. The followingsequence is employed: (1) fixed, (2) washed, (3) blocked, (4) cocktailwith markers (e.g., Hoechst 3341, LC-lectin, DM1-α, and a primaryantibody to the phosphorylated histone (pH3)), (5) wash, (6) contactwith a fluorescent secondary antibody to the pH3 primary antibody, (7)wash, and (8) image. Hoechst 3341 is a DNA marker, LC-lectin is a Golgimarker, and DM1-α is a tubulin marker.

As indicated, one can use the washout coefficient in determining a levelof apoptosis. Another indicator of apoptosis is a combination of (1) anda high mitotic percentage of cells with condensed DNA (using a DNAmarker) and (2) a low mitotic index using a phospho-histone marker(e.g., a pH3 marker). As indicated, during apoptosis, the nuclear DNAbecomes condensed and fragmented. Thus, proportion of cells withcondensed DNA will increase. Of course, a stimulus that produces mitoticarrest will also give a high mitotic index using a DNA marker. So, amere quantification of cells with condensed DNA using a DNA marker willnot provide strong evidence of apoptosis. To more clearly distinguishstimuli that induce mitotic arrest from stimuli that induce apoptosis,the procedure uses a marker (e.g., an antibody) for a phosphorylatedhistone, e.g., phospho-histone 3 (pH3). During mitosis, the histones inthe nucleus become phosphorylated. Therefore, mitotic index is measuredusing a pH3 marker will also give a high reading for the stimuli thatinduce mitotic arrest. However, during apoptosis, histones are notphosphorylated to such a degree. Therefore, a histone-based mitoticindex will not be so high for apoptotic cells. One view of thisdistinction may be understood with reference to the following table.Cells with Condensed DNA Mitotic Index (Histone) Mitotic Arrest High +High + Apoptosis High + Low −V. Detecting Steatosis

FIGS. 4A and 4B present cartoon representations of hepatocytesassociated with steatosis. Steatosis is a form of cytotoxicitycharacterized by a build up of triglycerides within hepatocytes. It isalso manifest by an accumulation of lipid containing vacuoles 14 withinthe hepatocyte cells 200. It is caused by inhibition of lipid transportaway from the liver or by inhibition of mitochondrial oxidation of fattyacids that normally participates in lipid breakdown. Accordingly, oneway to determine that a cell exhibits steatosis includes detecting thebuildup of lipids in the cell.

Steatosis can be microvesicular or macrovesicular. FIG. 4A shows acartoon representation of microvesicular steatosis. As shown, themicrovesicles 14′ of hepatocyte 200 become more numerous than in thenormal hepatocyte 12. In microvesicular steatosis, the numerous tinylipid droplets do not displace the nucleus 16. FIG. 4B shows a cartoonrepresentation of macrovesicular steatosis. Macrovesicular steatosisoccurs when larger lipid droplets 14″ are formed within hepatocyte 200′and displace the nucleus 16 to the periphery.

In one embodiment, lipid-binding markers are employed to detectsteatosis. In a specific example, the lipid-binding fluorescent probe,Nile Red (available from Molecular Probes, Inc., Eugene, Oreg.) isintroduced to in vitro cultures of hepatocytes exposed to a stimulus.Once the Nile Red has been introduced to the hepatocytes, thehepatocytes can be imaged. If a relatively large fluorescence signal isdetected from the images, then it can be inferred that the hepatocytesmay exhibit steatosis. In previous studies using the human hepatoma cellline HepG2, increased Nile Red binding was observed after the HepG2cells were incubated with compounds known to cause hepatic fataccumulation in vivo, including estrogen, ethionine, cyclosporin A, andvalproic acid. Image analysis can provide an indication of thelocations, sizes, and shapes of lipid vesicles within hepatocytes toprovide an additional indication of steatosis and whether it ismicrovesicular or macrovesicular.

An exemplary procedure for performing a Nile Red assay will now bedescribed. Nile Red (Molecular Probes, Inc., Eugene, Oreg.) is dilutedto 1:1000 in DMSO from a 1 mM stock solution and added to hepatocytes.The hepatocytes are then incubated for 2 hours in the dark. Thehepatocyte cells are then washed two times in PBS, fixed in 3.7%formaldehyde containing 1 μM Hoechst 33342 and a succinimidyl esterconjugated to a fluorescent dye. The plate is incubated for 1 hour inthe dark at room temperature. Plates are washed 3 times with PBS andimages are collected. As appreciated by those of skill in the art,modifications can be made to the assay within the scope of the presentinvention. Furthermore, alternative or additional assays can be used.

Alcohol, a common cause of steatosis, is a hepatotoxin that interfereswith mitochondrial and microsomal function in hepatocytes, which leadsto an accumulation of lipid. Also, the major toxic metabolite ofalcohol, acetaldehyde, has been shown to impair microtubulepolymerization and hepatic protein secretion. Thus, visualization ofmicrotubule morphology using, for example, DM1-α as a marker may providea useful assay for predicting hepatotoxicity caused by alcohol.

VI. Detecting Cholestasis

Generally, cholestasis is characterized as inhibition of bile flowcaused by a wide variety of mechanisms that involve elements of thebiliary tree, including bile ducts, ductules, the basolateral orcanalicular membrane, the tight junctions or pericanalicular network ofthe hepatocytes, the ATPase, and transporters of the hepatocytes'basolateral and canalicular plasma membranes. It may involve defects ofthe transport of bile acids from the sinusoidal blood into hepatocytesor from hepatocytes into bile. Thus, assays for cholestasis includeimaging of bile using light microscopy to directly measure theimpairment of bile flow.

Some drugs may cause cholestasis by damaging pericanalicularmicrofilaments. For example, cytochalasin B has been shown to produce aprompt arrest of bile flow in rats, thereby resulting in cholestaticinjury. In addition, phalloidin causes an increase in filamentous Factin around canaliculi and tight junctions. Thus, changes in actinmorphology may be indicative of cholestatic injury.

Further, cholestasis has been implicated in the disruption of tightjunction proteins. Hence, a method that images tight junctions canprovide insight into the cholestasis-inducing effects of a givenstimulus. One class of tight junction proteins that have been consideredfor this purpose is the zonula occludens-1 (ZO-1), ZO-2, and ZO-3. Othertight junction protein classes include claudin 1, 2, 3, and occludin.Fluorescent antibodies to ZO-1 can be used in cholestasis assays of thisinvention. Another marker for detecting cholestasis is the intermediatefilament protein cytokeratin, which is located in the pericanalicularregion of normal hepatocytes. During cholestasis, the pericanalicularsheeth is disrupted and the distribution of cytokeratin is altered.Thus, cytokeratin may be a useful marker for detecting cholestasis inhepatocytes.

VII. Creating Models for Hepatotoxicity

Models for hepatotoxicity can be generated using a variety ofmethodologies known to those of skill in the art. U.S. patentapplication Ser. No. 10/623,485 (“Characterizing Biological Stimuli byResponse Curves,” filed on Jul. 18, 2003), previously incorporated byreference, describes one such technique. It identifies relevant featuresand other parameters for image analysis classification models byanalyzing stimulus response paths for various known toxins or otherstimuli. The response paths and known mechanisms of action (orpathologies) comprise a training set for the model. Various potentialmodels are compared based on their ability to correctly classify membersof the training set. The classification is accomplished using distancemeasurements between the various stimulus response paths in amultidimensional feature space. Similar methods may be employed togenerate hepatotoxicity models in accordance with this invention.

Generally, the training set includes signatures or response paths orsignatures for diverse stimuli that have been previously classified by atrustworthy method. For example, the response paths may be obtained forstimuli having known toxicity (e.g., cholestasis, steatosis, necrosis,apoptosis, control, etc.). As explained above, the response paths arecomprised of (or derived from) signatures of the stimuli at variouslevels or various times.

In one example of a training set, toxins A-F are known to inducecholestasis, toxins G-K are known to induce steatosis, toxins L-P areknown to induce apoptosis in hepatocytes, compounds Q-T are known to bebenign to hepatocytes, etc. Hepatocyte cultures are exposed to each ofthese toxins/compounds and imaged using a defined marker set andtreatment regimen. Features extracted from the images comprise thetraining set for developing a hepatotoxicity model. The training setpreferably includes some negative controls. These may be derived fromstimuli that do not affect hepatocytes. They may also be derived fromcell lines that are relatively insensitive to particular toxins, incomparison to hepatocytes. Further, the training set may include imagesof cells undergoing toxic responses other than hepatotoxicity. Examplesinclude cardiotoxicity and neural toxicity.

The process of creating a model may identify specific features that areparticularly useful in classifying hepatocyte pathologies. The rawtraining set will include a large number of features extracted from manyimages collected from different experimental conditions: i.e. celltypes, compound concentrations, marker sets, etc. Multiple features fromeach image will be combined to produce a “signature,” a largemultidimensional vector comprised of values for each biological featurethat might potentially be employed in the model. So, in other words, theinitial data set (from the training set members) occupies a highlymultidimensional space defined by all available biological features. Atthe end of the process, only some of these features (a subset) will beselected for the hepatotoxicity model. Obviously, there will be a vastnumber of possible subset combinations. The model generating processchooses a combination (feature subset) that does a good job ofdistinguishing between the various pathologies.

The various members of the training set (as represented by therespective signatures or response curves) can be grouped or classifiedbased on distances in feature space. Various classification algorithmsmay be employed. Some are presented in “The Elements of StatisticalLearning, Data Mining, Inference and Prediction,” T. Hastie, R.Tibishirani, J. Freidman Springer 2001, which is incorporated herein byreference for all purposes. Examples include Nearest Neighbor methods,Linear Discriminant Analysis, and the like. If the training set is knownto contain stimuli that produce eight different hepatocyte pathologies,for example, the classification algorithm may be asked to segregate theresponse curves into eight different groups (for the current model underconsideration). The accuracy of this grouping is then assessed bycomparing the grouping to the known pathologies (or otherclassifications) for the training set members. Good models will haverelatively few misclassifications.

Note that models can be validated. Various validation techniques areknown to those of skill in the art. Some of these employ test sets thatdo not overlap with the training set but have known classificationsaccording to criteria such as mechanism of action, and the like. Othersemploy a “leave one out” technique in which the model is generated fromall members of the training set, except one. The resulting model is thenevaluated on the basis of its ability to properly classify the “leftout” member. This process can be repeated numerous times by choosingdifferent training set members to “leave out” during the modelgenerating operation

With relevant feature set identified, the model may be essentiallycomplete. To use the model one extracts the relevant features from animage of hepatocytes treated with a stimulus having unknown toxicity.Then one measures distances between these features and features obtainedfrom other stimuli having known toxic responses. Classification is basedon distance (in feature space) between the features of the test stimuliand features of the various pre-classified stimuli. Alternatively, onecan use a regression technique, a neural network, a support vectormachine, etc. To develop an expression or analytical tool that takesfeature values as input values and calculates a classification(pathology).

VIII. Database

According to various embodiments, a database is employed to assist incharacterizing the toxic response of various stimuli or classes ofstimuli. Preferably, the database is continuously monitored for qualityby testing its ability to predict the hepatotoxicity of new compounds inanimals and humans. In some embodiments, the database includes pathologydata from other tissues, such as kidney tissue.

In other embodiments, it is appropriate to diversify the data sets byincorporating other assay technologies. For example, gene expressiondata using RNA isolated from in vitro hepatocyte cultures or animalliver biopsies can be useful for predicting specific mechanisms oftoxicity that are difficult to detect using cell-based imagingapproaches. In addition, new biomarkers can be identified from the geneexpression data. These biomarkers may be useful in the cell imagingassays described above. For example, the gene products identified fromgenes specific to a steatosis response could be used to generateantibody reagents for the cell imaging assays. These antibody reagentscan be used as novel diagnostic regions to monitor the serum levels ofpatients during clinical trials for early signs of hepatotoxicity.

VIII. Software/Hardware

Generally, embodiments of the present invention employ various processesinvolving data stored in or transferred through one or more computersystems. Embodiments of the present invention also relate to anapparatus for performing these operations. This apparatus may bespecially constructed for the required purposes, or it may be ageneral-purpose computer selectively activated or reconfigured by acomputer program and/or data structure stored in the computer. Theprocesses presented herein are not inherently related to any particularcomputer or other apparatus. In particular, various general-purposemachines may be used with programs written in accordance with theteachings herein, or it may be more convenient to construct a morespecialized apparatus to perform the required method steps. A particularstructure for a variety of these machines will appear from thedescription given below.

In addition, embodiments of the present invention relate to computerreadable media or computer program products that include programinstructions and/or data (including data structures) for performingvarious computer-implemented operations. Examples of computer-readablemedia include, but are not limited to, magnetic media such as harddisks, floppy disks, and magnetic tape; optical media such as CD-ROMdisks; magneto-optical media; semiconductor memory devices, and hardwaredevices that are specially configured to store and perform programinstructions, such as read-only memory devices (ROM) and random accessmemory (RAM). The data and program instructions of this invention mayalso be embodied on a carrier wave or other transport medium. Examplesof program instructions include both machine code, such as produced by acompiler, and files containing higher level code that may be executed bythe computer using an interpreter.

FIG. 5 illustrates a typical computer system that, when appropriatelyconfigured or designed, can serve as an image analysis apparatus of thisinvention. The computer system 500 includes any number of processors 502(also referred to as central processing units, or CPUs) that are coupledto storage devices including primary storage 506 (typically a randomaccess memory, or RAM), primary storage 504 (typically a read onlymemory, or ROM). CPU 502 may be of various types includingmicrocontrollers and microprocessors such as programmable devices (e.g.,CPLDs and FPGAs) and unprogrammable devices such as gate array ASICs orgeneral purpose microprocessors. As is well known in the art, primarystorage 504 acts to transfer data and instructions uni-directionally tothe CPU and primary storage 506 is used typically to transfer data andinstructions in a bi-directional manner. Both of these primary storagedevices may include any suitable computer-readable media such as thosedescribed above. A mass storage device 508 is also coupledbi-directionally to CPU 502 and provides additional data storagecapacity and may include any of the computer-readable media describedabove. Mass storage device 508 may be used to store programs, data andthe like and is typically a secondary storage medium such as a harddisk. It will be appreciated that the information retained within themass storage device 508, may, in appropriate cases, be incorporated instandard fashion as part of primary storage 506 as virtual memory. Aspecific mass storage device such as a CD-ROM 514 may also pass datauni-directionally to the CPU.

CPU 502 is also coupled to an interface 510 that connects to one or moreinput/output devices such as such as video monitors, track balls, mice,keyboards, microphones, touch-sensitive displays, transducer cardreaders, magnetic or paper tape readers, tablets, styluses, voice orhandwriting recognizers, or other well-known input devices such as, ofcourse, other computers. Finally, CPU 502 optionally may be coupled toan external device such as a database or a computer ortelecommunications network using an external connection as showngenerally at 512. With such a connection, it is contemplated that theCPU might receive information from the network, or might outputinformation to the network in the course of performing the method stepsdescribed herein.

In one embodiment, the computer system 500 is directly coupled to animage acquisition system such as an optical imaging system that capturesimages of cells. Digital images from the image generating system areprovided via interface 512 for image analysis by system 500.Alternatively, the images processed by system 500 are provided from animage storage source such as a database or other repository of cellimages. Again, the images are provided via interface 512. Once in theimage analysis apparatus 500, a memory device such as primary storage506 or mass storage 508 buffers or stores, at least temporarily, digitalimages of the cell. With this data, the image analysis apparatus 500 canperform various image analysis operations such as classifying a cellaccording to a particular hepatotoxic pathology. To this end, theprocessor may perform various operations on the stored digital image.For example, it may analyze said image in manner that extracts values ofone or more descriptors and classifies the cell as alive or dead or asexhibiting apoptosis, necrosis, steatosis, cholestasis, or some otherpathology.

IX. Example Assays

To measure the viability of hepatocytes after plating, an aqueousCellTiter Non-radioactive Cell Proliferation Assay may be used (Promega,Madison, Wis.). This assay can also be used to measure the hepatoxicityof a test compound.

In one example of the assay, hepatoxicity is determined by assaying rathepatocytes for viability following a 24-hour exposure to a 9-pointdilution series of compound (to allow assessment of dose). Cellviability is determined by measuring the absorbance of formazon, aproduct formed by the bioreduction of MTS/PMS, commercially availablereagents. MTS is(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (Promega Corp. Madison, Wis.). PMS is phanazine methosulfate(TCI America, Portland, Oreg.). Each point on a dose-response curve iscalculated as a percent of untreated control cells at 24 hours minusbackground absorption (complete cell kill). Coumarin is specificallytoxic to rat hepatocytes under these conditions with an IC50 ofapproximately 53 micromolar.

Materials and Solutions:

-   Cells: Primary rat hepatocytes-   Media: Hepatozyme (Invitrogen)-   Colorimetric Agent for Determining Cell Viability: Promega MTS    tetrazolium compound.-   Control Compound for maximum cell kill: 0.1% Triton X-100-   Procedure:    Day 1—Cell Plating-   A. Prepare rat hepatocytes and seed into the wells of a 96-well TC    plate (Biocoat Collagen 1) at a density of 50,000 cells per well.-   B. Incubate with HBM or Hepatozyme media for 24 hours at 37° C.,    100% humidity, and 5% CO₂, allowing the cells to adhere to the    plates.    Day 2—Compound Addition

With reference to FIG. 6, in a sterile 2 mL v-bottom assay block,dispense 2.5 μl of compound at 400× highest desired concentration towells in column 3, rows B through G. (six different compounds per block,one for each row, edge wells are not used due to evaporation). Thecompound is used at 400× to ensure a final concentration of vehicle(DMSO) on cells is 0.25%. Next, add 250 μL of media containing 0.2%Triton X-100 to column 2, rows E, F, and G. ODs (optical density orabsorbance) from this well will be used to subtract out for backgroundabsorbance of dead cells and vehicle. Add 500 μl of media without DMSOto each well in column three, and 250 μL to wells E, F, and G in column2. Add 250 μL media+0.5% DMSO to all remaining wells. Serially dilutecompound 2-fold from column three to column eleven by transferring 250μl from one column to the next taking care to mix thoroughly each time.

-   A. By row, replica plate 100 μL 2× compound-containing media in    duplicate from the assay block to the corresponding cell plates.    Incubate plates for 24 hours at 37° C., 100% humidity, and 5% CO₂.    Day 3—MTS Addition and OD Reading-   A. After 24 hours of incubation with drug, remove plates from    incubator and add 40 μl MTS/PMS to each well. Incubate plates for    120 minutes at 37° C., 100% humidity, 5% CO₂. Read ODs at 490 nm    after a 5 second shaking cycle in a ninety-six well    spectrophotometer.

For Data analysis, one may calculate normalized % of control(absorbance-background), and use XLfit (ID Business Solutions Limited,Guildford, Surrey, UK). to generate a dose-response curve.

This assay was used to measure the toxicity of a training set of 28known hepatotoxins for primary rat hepatocytes, SKOV3 cells (an ovariancancer cell line) and CHO-K1 (Chinese hamster ovary cells). The resultsare shown in FIGS. 7A and 7B.

Cytochrome P450 enzymes are an important class of hepatocyte enzymesresponsible for drug metabolism. Their enzyme activity or geneexpression can be used to access the quality of plated hepatocytes.Various techniques can be employed to measure induction of specific P450genes in hepatocytes. For example, TaqMan, or branched DNA (bDNA)methods can be used. To measure the enzyme activity of rat cytochromeenzymes 1A1 and 1A2, the following fluorescence-based assay can be used(modified from Donato et al., Analytical Biochemistry, 213, 29-33(1993), which is incorporated herein by reference.)

-   1. Incubate cells with 100 μl Hepatozyme in the presence or absence    of 5 μM 3-methylcholanthrene (Sigma 213942) for at least 24 hours.    Control wells contain equivalent amount of vehicle (DMSO).-   2. Wash cells 1× with 100 μl PBS.-   3. To each well, add 100 μl PBS containing 10 μM ethoxyresorufin    (Molecular Probes R-352) and 10 μM dicoumarol (VWR IC10155525).    Store these compounds in DMSO and dilute just prior to use. Incubate    cells at 37 degrees for 30 min.-   4. Transfer 75 μl from each well of the assay plate to a 96-well    black plate (Costar 3915).-   5. Prepare a resourfin standard curve for the 96-well black plate.    Add 75 μl of a 2.6 μM resorufin (Molecular Probes R-363) is    equivalent to 200 pmoles of resorufin. Prepare a seven point    dilution series (2-fold).-   5. Add 25 μl containing 15 Fishman units of β-glucuronidase and 120    Roy units of arylsulfatase in 0.1M sodium phosphate buffer pH 4.5.    Dilute enzyme stock solution (Roche 127 698) 1:166 in phosphate    buffer. Shake plate.-   6. Incubate 1-2 hrs at 37 degrees to hydrolyze resorufin glucoronic    and sulfate conjugates.-   7. Add 200 μl of absolute ethanol to each well.-   8. Read fluorescence (530 nm ex. and 590 nm em.) using a Tecan    Safire plate reader (Tecan, Maennedorf, Switzerland). Optimize gain    using 200 pmole standard well. Number of flashes is 3 and    integration time is 40 microseconds. The plate format is GRE96fb.-   9. Calculate fluorescence and express activity as pmoles of    resorufin formed per well in 30 min.    X. Other Embodiments

The above discussion has focused on hepatocytes and hepatotoxicresponses. Aspects of the invention extend beyond hepatotoxicity totoxicity in a variety of other cell lines, cell types, and tissues.Further, it may be employed to classify various non-hepatocyte celltypes based on specific pathologies. Examples include myelosuppressionusing bone marrow cells, and nephrotoxicity using kidney cells.

Although the above generally describes the present invention accordingto specific exemplary processes and apparatus, various modifications canbe made without departing from the spirit and/or scope of the presentinvention. Therefore, the present invention should not be construed asbeing limited to the specific forms shown in the appended figures anddescribed above.

1. A method of assessing the hepatotoxicity of a stimulus, the methodcomprising: (a) analyzing an image of hepatocytes that have been exposedto a stimulus, wherein the analysis extracts features characterizing thehepatocytes; and (b) classifying the stimulus by quantitativelyevaluating the extracted features to identify one or more hepatotoxicpathologies resulting from the stimulus, wherein hepatotoxic pathologyclassifications include two or more of the following: necrosis,cholestasis, steatosis, fibrosis, apoptosis, and cirrhosis.
 2. Themethod of claim 1, further comprising, prior to analyzing the image:exposing a hepatocyte culture to the stimulus; and imaging thehepatocytes to produce the image.
 3. The method of claim 2 whereinmultiple hepatocyte cultures are located on a single support structure,and wherein each in vitro culture is exposed to a distinct stimulus. 4.The method of claim 3, wherein at least two of the cultures are exposedto different quantities of the same stimulus.
 5. The method of claim 3,wherein the support structure is a glass or plastic support.
 6. Themethod of claim 3, wherein hepatocytes are co-cultured with supportcells.
 7. The method of claim 1, wherein the stimulus is exposure to achemical compound.
 8. The method of claim 1, wherein the hepatocytes aretransformed or immortalized cells.
 9. The method of claim 8, wherein thetransformed or immortalized cells have been modified to express one ormore cytochrome P450 metabolizing enzymes.
 10. The method of claim 1,wherein analyzing the image comprises segmenting the image to identifyindividual hepatocytes on the image.
 11. The method of claim 1, whereinthe features extracted in (a) comprise two or more of membranepermeability, enzyme activity, Golgi distribution, migration ofcytochrome c from the mitrochondria, mitochondrial membrane potential,condensation, fragmentation and granularization of nuclei, accumulationof lipid containing vacuoles, bile production, actin morphology, andtight junction condition.
 12. A method of identifying a necrotichepatotoxic pathology resulting from a stimulus, the method comprising:(a) exposing a hepatocyte culture to the stimulus; (b) contacting thehepatocyte culture with markers for esterase activity and cell membranepermeability; (c) imaging the hepatocyte culture; (d) analyzing imagesof the hepactocyte culture to extract features relevant to necrosis; (e)identifying the average levels of esterase activity and cell membranepermeability for the hepatocyte culture based on the extracted features;and (f) characterizing the necrotic response of the hepatocyte cultureto the stimulus based on the average levels of esterase activity andcell membrane permeability.
 13. The method of claim 12, wherein thehepatocyte culture is characterized as necrotic if at least one of lowesterase activity and high cell permeability is identified.
 14. Themethod of claim 12, wherein the marker for esterase activity is calceinAM.
 15. The method of claim 12, wherein the marker for cell membranepermeability is ethidium bromide homodimer.
 16. A method of identifyingan apoptotic hepatotoxic pathology resulting from a stimulus, the methodcomprising: (a) exposing a first and second hepatocyte culture to thestimulus; (b) performing a single step preparatory treatment of thefirst hepatocyte culture, wherein the single step preparatory treatmentdoes not include washing the first hepatocyte culture; (c) performing amulti-step preparatory treatment of the second hepatocyte culture,wherein the multi-step preparatory treatment includes washing the firsthepatocyte culture; (d) imaging the first and second hepatocytecultures; (e) analyzing images of the first and second hepactocytecultures to extract features relevant to apoptosis; (f) identifyingcondensation of the nuclei, cell adhesion, and average caspase-3activity for the first and second hepatocyte cultures based on theextracted features; and (g) characterizing the apoptotic response of thefirst and second hepatocyte cultures to the stimulus based on thecharacteristics of the nuclei, cell adhesion, and average caspase-3activity.
 17. The method of claim 16, wherein the first and secondhepatocyte cultures are characterized as apoptotic if at least one ofcondensation of the nuclei, lowering of cell adhesion, and increasedcaspase-3 activity are identified.
 18. The method of claim 16, furthercomprising exposing at least one of the first and second hepatocytecultures to a marker for DNA.
 19. The method of claim 16, wherein celladhesion is characterized by a washout coefficient.